Method and Devices for Running Reactions on a Target Plate for Maldi Mass Spectrometry

ABSTRACT

A peptide or protein microassay method and apparatus in which a wide variety of chromogenic or fluorogenic peptide or protein substrates of interest are individually suspended or dissolved in a hydrophilic carrier, with aliquots of each substrate being deposited in an array or microarray of reaction loci, or “dots.” Each dot, therefore, provides an individual reaction vessel containing the peptide or protein of interest, to which a biological sample may be applied for assay purposes. The sample is applied to the array or microarray of dots by one of a variety of focused sample application techniques, including aerosolizing or misting of the sample, or target application of the sample, onto each dot without creating fluid channels between the dots which would cause cross-contamination. In additional aspects, the present invention provides methods of transferring samples from an electrophoretic gel to a target plate for subsequent MALDI MS analysis. Chemical reactions of interest can be run directly on the target plate, and the reaction products on the target are then prepared for MALDI MS analysis by drying and aerosol deposition of matrix material, without the need for salt removal and additional processing steps.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/529,643 filed Dec. 15, 2003, and to U.S. Provisional PatentApplication Ser. No. 60/614,145 filed Sep. 29, 2004, and is also aContinuation-In-Part of U.S. patent application Ser. No. 10/036,066filed Nov. 7, 2001, which claims priority to each of the following U.S.(U.S.) Provisional Patent applications: U.S. Provisional PatentApplication Ser. No. 60/266,042 filed Feb. 2, 2001; U.S. ProvisionalPatent Application Ser. No. 60/309,999 filed Aug. 3, 2001; U.S.Provisional Patent Application Ser. No. 60/313,380 filed Aug. 17, 2001;U.S. Provisional Patent Application Ser. No. 60/313,368 filed Aug. 17,2001; U.S. Provisional Patent Application Ser. No. 60/313,377 filed Aug.17,2001; and U.S. Provisional Patent Application Ser. No. 60/322,619filed Sep. 17, 2001, each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a microassay chip and method for analysis bymeans of peptides or proteins for use in biological research andbiomedical diagnosis. In an additional aspect, the invention relates tomethods of transferring molecules of interest from an electrophoreticgel directly to a target plate for MALDI mass spectrometry analysis. Ina further aspect, methods for running chemical reactions on a targetplate, methods of preparing samples for MALDI mass spectrometryanalysis, and methods of depositing MALDI matrix on a target plate arealso included.

2. Description of Related Art

In biological research, biomedicine and industrial applications, largescale genomic evaluation for the detection of specific genes or DNAsequences within a genome, specific gene mutation such as singlenucleotide polymorphisms (SNP), and mRNA species are well-establishedmethodologies. These methodologies utilize DNA chips and microarrays onwhich specific nucleic acid sequences are either synthesized ordeposited at individual highly localized positions on an array. Thesearrays containing the nucleic acid sequences find support on solids suchas silicon or glass, or materials such as nylon membranes. The sequencescan exist in the array on the order of 10³ or 10⁴ individualmicrosamples because individual “dots” or “pixels” have sub-millimetercharacteristic lengths. While these chips have many applications fordetecting the presence of and identifying genes in a genome (genotyping)or evaluating patterns of gene regulation (mRNA profiling) in cellularand tissue systems, these nucleic acid-based systems provide noinformation about the activity or regulation of the gene product, i.e.,the synthesized protein.

Currently, DNA chips and microarrays allow genotyping and expressionprofiling, without rendering information about the activities of enzymeswhich can be regulated by phosphorylation or cleavage states. Proteinchips to date have involved the capture of proteins to immobilized DNAsequences or libraries of immobilized peptides, antibodies or proteins.The three major formats for protein arrays employ plain glass slices,three-dimensional gel pad chips (“matrix” chips) or nanowell chips. Noneof these formats utilizes soluble substrates to identify numerousenzymes in a simple assay, however.

Proteomic methods typically utilize two-dimensional electrophoresis gelsto separate proteins, followed by enzyme digest mapping and/or massspectrometry to characterize relevant individual proteins in the gel.Neither DNA chips nor two-dimensional electrophoresis provideinformation about the activity of the protein or its reaction kinetics.For example, an enzyme may require phosphorylation or dephosphorylationin order to have full activity, and prior chip technologies do notprovide this information.

Presently, enzyme activity can be measured by incubation of the enzymewith chromogenic substrates whose cleavage products become intenselycolored and absorb light at a particular wavelength. Alternatively, thesubstrate may be a fluorogenic substrate whose cleavage results inleaving groups that are intensely fluorescent when excited at aparticular wavelength (8-EX). Emission wavelengths of the leaving groupsmay span 10 to 20 nm above and below the maximum 8-EM. This prevents theuse of more than two or three different fluorogenic substrates in asingle sample to assay for three different enzymatic activities sincethe emission of each substrate may have significant overlap with theemission of the other substrates. Broad band emission results in colorcross-talk and can render false signals. Thus, it is not possible to add10 to 100 different fluorogenic substrates to a single fluid samplebecause the emissions would overlap severely. These reactions aretypically monitored in cuvettes in a fluorimeter or plate-reader withworking volumes of 0.2 to 3 ml. Thus, significant dilution of the sampleoccurs.

The evaluation of various proteins and/or enzymes within a smallbiological sample (1.0 to 100 nL) would be useful in analyzing theactivity of those proteins and/or enzymes in a number of fields ofstudy. In the field of cell biology and cancer, the timing of celldivision is regulated by numerous cyclin-dependent kinases (cdk),cAMP-dependent kinases (PKA), cGMP-dependent kinases (PKG), andcalcium-dependent protein kinases (PKC), tyrosine kinases, and tyrosinephosphatases. In the field of hematology, the function of blood isregulated by various coagulation factors, complement factors andfibrinolytic factors which are proteases and inhibitors necessary forthrombotic and thrombolytic mechanisms. During apoptosis (programmedcell death) various caspases are critical to the cascade of events.Similarly, neutrophil activation during sepsis, thrombosis or infectionis coordinated with release of elastases, proteases or other enzymes.Tumor invasion and intimal hyperplasis can involve the activity of metalmetalloproteases (MMPs) and tissue inhibitor of metalloproteases(TIMPs). Various viral activities (e.g., proteases) would be suitablefor detection of drug screening of protease inhibitors.

Notwithstanding prior art developments in the areas of peptide andprotein chips, therefore, the need for peptide or protein microarrays indiagnostic, prognostic and clinical medicine is large, and largelyunmet. Prior art chips do not exist in which a great variety ofsuspended or soluble chromogenic or fluorogenic substrates may be simplydeposited in an array on a support surface, with simple application ofthe sample fluid thereto for evaluation. At this writing, there are noknown peptide or protein chips which can be directly fabricated using astandard contacting or non-contacting microarrayer, for example. Liquidlayer sample applications over unbound substrate molecules would beconsidered unthinkable, moreover, due to the inevitablecross-contamination such liquid sample layers would engender. As aresult, a need remains for a simple, effective and inexpensive peptideor protein array or microarray system which provides an easilyfabricated chip using standard microarrayer equipment, which provides asystem in which elaborate compensations such as peptide or proteinbinding, or quenching layers are unnecessary, and to which sample may besimply and easily applied. Also, the need likewise persists for a systemwhich can rapidly deliver small liquid samples to individual reactantpositions of an array or microarray without cross-contamination amongthe reactant positions.

Proteomics and high throughput screening (HTS) are activities thatinvolve the analysis of hundreds to millions of samples. In drugscreening, reactions are run in well-plates to produce optical signals(fluorescence, luminescence) indicating that a hit was identified. Thesearch for label-free drug screening could rely on matrix assisted laserdesorption/ionization (MALDI) mass spectrometry (MS), which hasexcellent throughput. However, the reaction constituents then have to beprepared for MS, in a series of steps which are time consuming andexpensive and limit the use of MALDI for HTS.

A common approach in proteomics research is to subject a complex proteinsample (a cell or tissue lysate) to separation by 2-dimensional gelelectrophoresis separation. Positions of high protein concentration, asindicated by dye staining are then removed mechanically as bands orplugs from the gel. The gel containing the sample is crushed to dispersethe sample and then subjected to a separation technique to remove theliquid containing the protein solutes. Proteins in the sample can besubjected to chemical cleavage or proteolytic degradation (typicallytrypsin) to create smaller fragments suited for mass spectrometry. Saltions are removed from the liquid sample using an ion-exchange resin. Thesample is ready for mixing with a MALDI matrix and then delivered bypositive displacement liquid handling to a position on a MALDI target.The sample is allowed to dry and is ready for interrogation by massspectrometry.

U.S. Pat. No. 5,808,300 (Caprioli, “Method and Apparatus for ImagingBiological Samples with MALDI MS”) discloses a method of depositingMALDI matrix material on a tissue sample by electrospraying a solutionof the matrix onto the sample.

U.S. Patent Publication 2002/0195558 discloses the use of acousticejection methods to deposit the MALDI matrix material on a tissuesample.

U.S. Pat. No. 6,288,390 (Siuzdak, “Desorption/Ionization of analytesfrom porous light-absorbing semiconductors”) teaches the use of poroussemiconductors for matrix-free mass spectrometry to replace conventionalMALDI. U.S. Pat. No. 6,288,390 is an alternative technology foranalyzing samples without the need for matrix materials.

U.S. Pat. No. 6,569,383 (Nelson, “Bioactive chip mass spectrometry”)relies on the capture (binding via biological affinity or chemicallinkage) of the analyte to the surface of a target.

Caprioli, R. M., J. Mass Spectrometry 38:1081-1092 (2002) discloses aprotocol for spraying matrix over a tissue sample by placing ˜20 ml of amatrix solution into a glass reagent sprayer Kontes Glass Company,Vineland N.J. USA) and spraying multiple coats of matrix across thesurface of the tissue. No mention is made of the use of the method fordrug discovery reactions on a MALDI target, and the disclosed methoddoes not describe use of an ultrasonic nozzle.

Caprioli, R. M., Electrophoresis 23, 3125-3135 (2002) describes anaerosol deposition coating method on to tissue. Matrix is air-sprayed onthe section using a commercially available glass spray nebulizerconnected to a nitrogen bottle (nebulizing glass) to minimizecontamination. No mention is made of the use of this method for drugscreening reactions.

There is a continued need for improvements in sample processing to highthroughput preparation and screening of samples using MALDI MS analysistechniques. Conducting HTS on a MALDI target would meet industry demandsfor label-free HTS with high capacity (10K to 100K screens per day).

SUMMARY OF THE INVENTION

In order to meet this need, the present invention is a peptide orprotein microassay method and apparatus in which a wide variety ofchromogenic or fluorogenic peptide or protein substrates of interest areindividually suspended or dissolved in a hydrophilic carrier, withaliquots of each substrate being deposited in an array or microarray ofreaction loci, or “dots.” Each dot, therefore, provides an individualreaction vessel containing the peptide or protein of interest to which abiological sample may be applied for assay purposes. The sample isapplied to the array or microarray of dots by one of a variety offocused sample application techniques, including aerosolizing or mistingof the sample, or target application of the sample, onto each dotwithout creating fluid channels between the dots which would causecross-contamination. In a first embodiment of the present invention, thesample is misted or aerosolized, and the application of such anaerosolized sample to the dots results in the sample's being absorbed bythe individual dots while any excess sample droplets between the dotseither tend to migrate toward and be absorbed by the nearest dot, orevaporate, leaving each dot as a discrete reaction chamber without fluidreactant connection to any other dot. Known scanning and databasecreation techniques may be used to analyze reaction indicators presentor absent in the arrays of dots.

The present invention provides methods of HTS and preparing samples forMALDI mass spectrometry analysis. Solutes within a solution are analyzedvia MALDI with no chemical linkage, immobilization, or adsorption to thesurface. Any changes to the solutes (via drug screening reaction,chemical reaction, or enzymatic reaction) occur in the solution phaseand do not require linkage of the components to the surface.

The present invention allows HTS reactions to be run directly on a MALDIplate followed by MALDI MS analysis with a detection limit of 1femtomole of a peptide in 50 nanoliters of sample. Methods of thepresent invention employ the power of contact pin printers to load MALDIplates at up to 1000 drops per square centimeter. Using the methods ofthe present invention, MALDI matrix can be applied with high qualitysuch that ultrasmall liquid samples can be used while still generatingoutstanding crystals for the MALDI process to allow ultrasensitivedetection.

The methods of the present invention provide the ability to runindividual nanoliter to microliter volume liquid reactions at positionson a mass spectrometry (MS) target plate. The target is a non-porousmetal surface that is flat or has wells, coatings, grooves or othermeans to maintain sample position. Reagents are added to each sampleposition by various liquid handling protocols to initiate reactions. Theconstituents and products of each reaction are then prepared for MALDImass spectrometry analysis by drying and deposition of the MALDI matrix,without the need for removal of the sample from the MS target forvarious manipulations, such as desalting, extraction, solvent exchange,digestion, MALDI matrix addition and formation. These methods can beused for reaction optimization, high throughput drug discovery, highthroughput drug selectivity profiling or toxicity testing.

More specifically, the present invention provides a method oftransferring molecules of interest from an electrophoretic polymer gelto a MALDI target plate comprising the steps of:

-   -   (i) providing an electrophoretic gel containing one or more        molecules of interest;    -   (ii) replacing water within the electrophoretic gel with a        cosolvent mixture;    -   (iii) positioning a pin over the gel and penetrating the gel        with the pin;    -   (iv) energizing the pin to deplete the gel in a region        surrounding the one or more molecules of interest, causing the        cosolvent mixture to surround the one or more molecules of        interest;    -   (v) lifting the pin out of the gel, the pin carrying a drop of        the cosolvent mixture containing the one or more molecules of        interest; and    -   (vi) contacting a MALDI target plate with the pin, the        contacting causing the drop of cosolvent mixture containing the        one or more molecules of interest to be deposited on the MALDI        target plate.

In a further aspect, the present invention provides a method of runningchemical reactions on a MALDI target plate comprising: depositing dropsof reactants on the target plate; depositing a reagent on the targetplate such that the reagent contacts the deposited drops; and allowingthe chemical reaction to proceed.

In yet a further aspect, the present invention provides a method ofpreparing a sample for MALDI mass spectrometry analysis comprising thesteps of:

-   -   (i) providing a target plate having liquid drops of sample;    -   (ii) drying the target plate to remove solvents from the sample        drops;    -   (iii) depositing a MALDI matrix onto the dry target plate;    -   (iv) humidifying the target plate; and    -   (v) subjecting the target plate to MALDI mass spectrometry for        analysis of the sample drops.

In an additional aspect, the present invention provides a method ofdepositing one ore more layers of a MALDI matrix on a target platecomprising:

-   -   (i) providing a target plate having samples thereon;    -   (ii) aerosolizing the matrix; and    -   (iii) spraying the aerosolized matrix on the target plate while        moving the target plate.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1 is a partial perspective view of an array according to thepresent invention;

FIGS. 2A, 2B and 2C are side elevational views of arraying, aerosolsample deposition and substrate conversion, respectively;

FIGS. 3A and 3B are schematic diagrams of peptide or protein microarraysbefore and after sample application;

FIG. 4 is a sectional view of an ultrasonic misting device;

FIG. 5 is a functional diagram of the assay apparatus;

FIG. 6 is an array of carrier solvent (glycerol) microdots;

FIG. 7 is a prespray array of carrier solvent (glycerol) microdots;

FIG. 8 is an array of microdots sprayed with water-based sample;

FIG. 9 is a proximal view of a sprayed microdot array after mistevaporation;

FIG. 10 is an activated microdot array;

FIG. 11 shows fusion of water droplets containing sample with glycerolmicrodot;

FIG. 12 shows detection of thrombin activity by microdot assay;

FIGS. 13A, 13B, 13C, and 13D show microfluidics technology for reagentdelivery to individual reaction compartments;

FIG. 14 shows delivery of molecules to reaction spots using spraygenerated from an ultrasonic nozzle;

FIG. 15 shows generation of ultrafine mist using ultrasound transducerand non-contacting chamber;

FIG. 16 shows delivery of molecules to reactive spots using smalldroplet spray generated by an ultrasound transducer and non-contactingchamber;

FIG. 17 shows that adjacent reaction spots do not cross-contaminateafter spray delivery of mist;

FIG. 18 shows a microarray assay of purified enzymes and human plasma;

FIG. 19 is a microarray of caspase substrate;

FIG. 20 is an activated caspase microarray;

FIG. 21 shows mist delivered by an assay system;

FIG. 22 shows fluorescent mist delivered to microarray; and

FIG. 23 shows capture of mist on microarray using an electrostaticcharge.

FIG. 24 is a schematic diagram illustrating a method transferringmolecules of interest from an electrophoretic gel to a MALDI targetplate.

FIG. 25 is a schematic diagram illustrating methods of running chemicalreactions directly on a target plate and then preparing the target platefor MALDI mass spectrometry analysis.

FIGS. 26 A-B are computer-generated drawings and mass spectrum dataobtained from the analysis as described in Example 10.

FIGS. 27 A-O are mass spectrum data obtained from the analysis asdescribed in Example 11.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

The present invention is a peptide or protein microassay method andapparatus in which a wide variety of chromogenic or fluorogenic peptideor protein substrates of interest are individually suspended ordissolved in a hydrophilic carrier, with aliquots of each substratebeing deposited in an array or microarray of reaction loci, or “dots.”Each dot, therefore, provides an individual reaction vessel containingthe peptide or protein of interest to which a biological sample may beapplied for assay purposes. The sample is applied to the array ormicroarray of dots by one of a variety of focussed sample applicationtechniques, including aerosolizing or misting of the sample, or targetapplication of the sample, onto each dot without creating fluid channelsbetween the dots which would cause cross-contamination. In a firstembodiment of the present invention, the sample is misted oraerosolized, and the application of such an aerosolized sample to thedots results in the sample's being absorbed by the individual dots whileany excess sample droplets between the dots either tend to migratetoward and be absorbed by the nearest dot, or evaporate, leaving eachdot as a discrete reaction chamber without fluid connection to any otherdot. Known scanning and database creation techniques may be used toanalyze reaction indicia present or absent in the arrays of dots.

Generally, this invention can be applied to areas of biotechnology andbiomedicinal research. More specifically, this invention can be used tostudy enzyme activities, as well as cofactors, inhibitors and activatorsof enzymes. In one application, the assay may be used in drug research,namely drug discovery, by screening the effect of large combinatoriallibraries of compounds on activities of generally between ten up tothousands of enzymes, or drug interaction in blood chemistry, matrixmetalloproteases, angiogenesis, tyrosine phosphoprotein phosphatases, orapoptosis regulation. The assay can be applied to genomics or proteomicsresearch, in particular, epigenetic regulation of enzyme systems. In afurther application, the assay can be used to research signaltransduction pathways, such as kinase and phosphatases in generegulation. The assay may also be used for structural and functionalresearch of combinatorial studies involving point mutations on enzymesubstrate specificity. The assay may be applied to blood research,namely, coagulation diagnostics and thrombolytic research, or the assaycan be applied to viral research and diagnostics of viral proteases andprocessing activities. The assay can thus be employed throughout variousfields of biological research due to its simplicity and versatility. Asmentioned above, the assay is suitable for use with existing scanningtechnologies in place for genomic studies.

In the above and ensuing description, the following terms may beunderstood as follows. A reaction loci is generally an adherentnon-spreading volume of fluid on a solid surface. A reaction spot can bereferred to as “spot”, “dot”, “reaction zone”, “reaction center”,“microdot”, or “microassay.” A chip is a planar surface containingnon-spreading reaction dots. Chips can also be referred to as a “glassslide”, “slide”, “surface”, “solid substrate”, “bioreaction microarray”,“bioreaction chip”, and “bioreaction slide.” Spray refers to thedelivery of an aerosol of liquid sample to a solid surface containingreaction spots. Spray can also be referred to as a “mist”, “aerosol”,“atomized mist”, “droplet/s” or “nebulized mist.”

The present assay generally comprises microreactions in a liquid phasewhich are created by applying small volumes of a fluid mixture of apeptide or protein substrate, a hydrophilic carrier solvent and avolatile solvent to a nonporous surface, whereby evaporation of thevolatile solvent results in highly localized long-lasting liquid orsemi-solid dot or microdot residues of substrate in a hydrophiliccarrier solvent. The substrate is fluorogenic or chromogenic to enableanalysis of the reaction, if any, within the hydrophilic carrier afterthe sample is applied. The nonporous surfaces for delivery of the fluidmixture can include silicon, glass, silica, quartz, polystyrene or othernonporous polymeric membranes. Overall, the components of the assay areusually combined and applied via a computer-controlled applicationsystem, and microreactions are monitored via a computer-based scanningand database producing system.

Particularly, when the arrays involved are microarrays, the presence ofthe volatile solvent facilitates fluid creation of the microdot byreducing the overall viscosity of the formative fluid admixture. Thevolatile solvent generally has the ability to evaporate and suitablevolatile solvents include, without limitation, dimethylsulfoxide (DMSO);chloroform; acetone; acetic acid; water; an alcohol such as methanol,ethanol or propanol; ethyl ether or alkane. After application of thefluid admixture to a nonporous surface, the volatile solvent evaporates,leaving microdots containing hydrophilic carrier solvent and thesuspended or dissolved chromogenic or fluorogenic substrate(s). Theseconstituents remain in a liquid or semi-solid state withoutcrystallization or precipitation of the substrate(s).

At the time of a sample application, the hydrophilic carrier suspends ordissolves the substrate(s) to maximize the bioreaction potential withlater applied biological samples. The hydrophilic carrier generallypossesses the following characteristics: miscibility with the volatilesolvent; miscibility with water; miscibility with aqueous biologicalfluids; suitability for maintaining a stable solution or suspension offluorogenic or chromogenic substrate(s) at high concentrations; moderateviscosity between 1 centipoise and 10,000 centipoise; compatibility withbiological molecules such as nucleic acids, peptides, proteins, andsugars; suitable fluidity for movement into and out of microcapillarydevices such as the hollow tips of microarray pins or microsyringes usedfor arraying; a specific contact angle sufficient to form a stablefinite lens where the bioreaction fluid in the spot after arraying doesnot spread (contact angle>0 is required); a specific contact angle lowenough to form a stable adherent lens that does not have too low ofadhesion such that the spot has limited adhesion and can roll on thesubstrate (contact angle<90 is required); and low volatility such thatthe reaction zone does not evaporate. Glycerol (1,2,3-propanetriol) isan example of such a fluid that possesses all of these characteristics.Other examples of the hydrophilic carrier solvent include a polyalcoholsuch as 1,2-ethanediol or 2,3-butanediol. In addition, the carriersolvent may contain viscosity enhancers such as dextran, pluronic acid,carbohydrates of the pentose, ribose or hexose families or relatedpolysaccharides or polyethylene glycol polymers.

The microdots are generally applied to the nonporous surface in amicroarray configuration. The final volume of the microdot, afterevaporation of the volatile solvent, ranges from about 1 nL for a 10 μmdiameter microdot to about 1 to 10 nL for a 100 μm dot. Microdots can beapplied through fluid handling methods of direct positive displacementpumping. Alternatively, the microdot is applied through “arraying”,whereby computer controlled metal, glass or plastic tips pick updroplets of fluid from a reservoir by capillary action and make contactwith the solid surface, or by laser printing or jet printing techniques.Arraying is accomplished by using well-established pin technologies(i.e., Telechem Pins, GeneMachine arrayer). The separation distancebetween microdots ranges from 50 to 1000 μm. Delivery of 1 to 10 nL offormulation is sufficient to create a microdot.

After creating a high density array of microdots, each of which containsa specific fluorogenic or chromogenic reporter substrate, as well asother possible reaction modifiers, a small sample of biological fluid isapplied to the microdots. Each microdot is inoculated with sample byapplication of the biological fluid, generally through deposition of afine mist on the biochip. The mist is applied in a manner that does notform a wetting film and never bridges two adjacent glycerol droplets. Inother words, the application of the aerosolized sample to the dotsresults in the sample's being absorbed by the individual dots while anyexcess sample droplets between the dots either tend to migrate towardarid be absorbed by the nearest dot, or evaporate, leaving each dot as adiscrete reaction chamber without fluid reactant connection to any otherdot. Delivery of the biological fluid containing a correspondingrelevant enzyme will cause reaction and concomitant activation of thechromogenic or fluorogenic substrate in each glycerol droplet. In otherwords, enzyme or chemical constituents of the biological fluid lead tothe activation or antagonism of the activation of the fluorogenicsubstrate in the microdot to produce a fluorogenic or chromogenic signalreadable by epifluorescence or confocal scanning, direct imaging orlight absorption. An individual chip can be configured to report theactivity of numerous proteases, kinases, phosphatases, oxidoreductases,lipases and inhibitors or activators of these enzymes, each within anindividual dot or microdot loci of each reaction of interest.

It should be borne in mind that the present peptide and protein chipsare considerably simpler than most if not all prior art arrangementswhich include means for physically adsorbing or binding peptides orproteins directly to the glass slide or chip, or which contain multiplecomponents including but not limited to quenching overlayers, gel padsor other features more complex than the present reaction loci.Constituents inconsistent with the practice of the present inventionwould be anything which would interfere with the hydrophilic carrier'sproviding a discrete reaction vessel containing the peptide or proteinof interest and any other constituents designed to facilitate sampleabsorption, reaction and reaction detection.

The invention is further illustrated in the accompanying FIGS. 1-5.

Referring now to FIG. 1, a biochip 10 has arrayed thereon a plurality ofreaction loci 12, over which are applied the aerosolized or misted orink-jet printed sample droplets 14. The vertical arrow illustratesvertical deposition of the sample droplets 14.

FIGS. 2A, 2B and 2C are side elevational views of arraying, aerosolsample deposition and substrate conversion, respectively, according tothe present invention. In FIG. 2A, a microarrayer tip 16 is showndepositing the reaction loci 12 onto the biochip 10. In FIG. 2B, theaerosolized or misted sample droplets 14 are shown in the process ofdeposition onto the reaction loci 12. In FIG. 2C, the sample droplets 14have either absorbed into the reaction loci 12 or have evaporated anddisappeared completely from the biochip 10. In FIG. 2C, individualreaction loci 12 can generate color or fluorescence as a result ofsubstrate conversion.

FIG. 3A is a schematic diagram showing a blanket sample application overall the reaction loci 12 of the biochip 10 in a square or rectangularpattern 18, whereas the reactant of FIG. 31B reposes solely within theconfines of the reaction loci 12 of the biochip 10. The result shown inFIG. 3B can occur through various mechanisms including but not limitedto the blanket sample application may be of an aerosol or mist whichevaporates from between the reaction loci 12; or the sample may betargeted for application to the reaction loci 12, such as by alaser-printer or ink-jet printer; or the sample may be applied through amask or template which blocks sample application from any area otherthan the reaction loci 12.

FIG. 4 illustrates, in section, a nebulizer 20 containing an ultrasonicgenerator (transducer) 21 filled with a liquid 22 adapted to receive acontainer 25 having a sample 26 therein, whereupon energization of thesample 26 by the transducer 21, the aerosol mist made from the sample 26may exit the nozzle 32 for deposition on the biochip 10. Optionally, acarrier gas 24 enters the container via inlet 23, which carrier gas 24helps to displace the aerosolized sample 26 for deposition onto thebiochip 10. For the purpose of FIG. 4, it should be borne in mind thatthe biochip 10 is inverted compared to the biochip position shown in theremaining Figures. The nebulizer 20 also contains optional placementguides 28 for holding the container 25 in position, as well as a cap 30for the container 25 through which the nozzle 32 extends as shown.

FIG. 5 is a schematic illustrating the juxtaposition of variouscomponents of the present assay system as positioned adjacent thebiochip 10, the reaction loci 12 and the sample droplet(s) 14. Thesecomponents may include, without limitation, a printhead for sampleapplication, an xyz positioner, a power supply, a controller, and meansfor providing excitation light and detecting emission signal, amongother components. More particularly, major components of the assaysystem (apparatus) include a set of operating instructions resident incomputer software that sends via serial or parallel port signals tostart, to stop, to establish operating set point, and to control thesubcomponents of the assay device, whereby each subcomponent may have aninternal or external standing controller or driver. The subcomponents ofthe device include the following: multiple positive displacementmicrosyringe pumps; aerosol generating devices, such as pressurenozzles, ultrasonic nozzles ink-jet printheads, position-actuatedink-jet printheads, surface-actuated ink-jet printheads, fluidcontacting or fluid non-contacting ultrasonic transducers; gas flowmeter/controller; xy positioner system; and an exhaust/filtration fan.

Aerosolized sample application may be facilitated through the use ofcomputer controlled microsyringes. The computer controlled microsyringesare used for timed sample delivery at constant low rate, and eachpositive displacement syringe can hold from 1.0 μL to 1000 μL ofbiological sample, whereby the sample may be organic molecules,fluorogenic molecules, peptides, proteins, lipids, dilute solutions ofpolymers, liquid with coated microbeads, sample buffers, wash bufferbiological cells and cell fractions. Each positive displacement pumpdelivers the sample to the site of aerosol generation. The positivedisplacement pump may be maintained in an environment that isrefrigerated, at room temperature or heated. The positive displacementpump is controlled by a device that receives signals from a computer.

There are various ways to aerosolize the sample for application to thepresent system. One system relies on pressure nozzles, whereby the fluidsample to be aerosolized is pumped at high pressure through smallorifice nozzles to generate an aerosol spray. In some nozzles, the fluidis carried by a pressurized inert carrier gas such as nitrogen, helium,air or oxygen. High velocity gas streams can be used to pull the fluidsample, by Bernoulli effect, into the nozzle and through the nozzleorifice. The carrier gas next delivers the aerosol to the biochip fordeposition on the individual reaction dots. Nozzles may have internalcomponents to facilitate aerosol formation. In one example, a smallbiological sample (0.1 to 1 ml) is pulled into a nozzle by pressurizedgas flow (5 to 30 psig nitrogen) through a 250 micrometer orifice with a210 micrometer inner needle to facilitate atomization.

Alternatively, spray aerosolization results from the use of ultrasonicnozzles which give low volumetric flow rate and uniform low velocity(under 10 cm/sec) mists. Ultrasonic standing waves within the nozzlecause atomization of the fluid at the tip of the nozzle. Low flow ratesfrom 0.01 to 1000 μL/sec can be achieved by micropump delivery of sampleinto the nozzle body that contains a piezoelectric ultrasound transduceroperating between 25 kHz to 240 kHz to create mists with average dropsizes from 5 to 15 micrometers in diameter. Energizing of thepiezoelectric ultrasound transducer can utilize low wattage (from 0.1 to25 watts) to avoid unwanted heating of the sample. For instance, abiological sample with the viscosity of 0.01 poise is pumped by amicrosyringe pump at a flow rate of 0.1 to 1 μL/sec into an ultrasonicnozzle operating at 120 kHz (0.1 to 1 watt). Carrier gas streamsexternal to the nozzle help to direct the mist to the bioreaction chipsurface.

Another means of creating aerosolized sample relies on a contactingultrasonic nebulizer where a fluid is placed in a well, the bottom ofwhich contains an ultrasonic piezoelectric transducer. The transducer isoperated at 1.0 to 3.0 MHz. The high frequency vibration at the topsurface of the liquid in the sample chamber facilitates the formation ofan atomized or nebulized cloud of fluid droplets. The action ofnebulization causes the nebulized aerosol to rise from the chambertoward a bioreaction chip surface suspended atop the chamber.Additionally, a carrier gas can be introduced into the nebulizingchamber upwardly to displace the cloud. Alternatively, a carrier gas canbe passed over the nebulizing chamber to pull the nebulized aerosol intothe carrier stream by Bernoulli effect. The carrier gas is directedtoward the samples to receive the aerosol. Atomized fluid particlediameter (d) is related by the surface tension (T), density (p), and thefrequency (f) by the following approximate equation of:d˜(T/pf³)/^(1/3). For example, nebulization of water (T=0.0729 N/m,f=2.4 MHz) produces 1.7 micrometer mist droplets.

A further means of aerosol generation relies on a non-contactingultrasonic nebulizer, in which a fluid to be delivered is placed in atube that has a thin walled plastic bottom suitable for transmission ofultrasonic waves. The tube is placed in a conducting fluid that is incontact with the ultrasonic transducer and the fluid sample to beaerosolized, therefore, never comes in contact with the ultrasonictransducer per se. The transducer is generally operated between 1.0 to3.0 MHz, and the high frequency vibration at the top surface of theliquid in the sample tube facilitates the formation of an atomized ornebulized cloud of fluid droplets in the sample tube. The action ofnebulization can cause the nebulized aerosol to rise in the chamber anda carrier gas is delivered into the sample tube to displace the mist(optionally through a coarse collecting filter to remove large droplets)toward the bioreaction chip. Nebulized fluid samples prepared with thisnon-contacting ultrasonic nebulizer will generally have droplet sizesranging from 1 to 25 micrometers.

Finally, ink-jet Piezo-printing, where delivery of a biological fluidinto the printing head that exploits non-heating ink-jet technology maybe used to propel the sample fluid toward a bioreaction spot. In thefirst approach of Piezo-printing, the printhead continually “prints” thebiological fluid over the entire slide in a raster pattern of printingwithout discrimination of its position over the reaction spot or overglass. In this non-specific mode of operation, the inkjet functions as aspray head, albeit one that creates a very narrow zone of spray and onethat requires xy positioning with time over the bioreaction slide inorder to actuate the entire slide. In this approach, the novel reactionzone isolation of the formulation fluid (glycerol) and the ability toprint without forming a wetting film or a continuous layer of printedbiological fluid allows reaction compartmentalization withoutspot-to-spot cross-bridging even though the entire surface is printed.

In the second approach of Piezo-printing, the printhead deliversbiological fluid at discrete times. The delivery of a droplet of fluidto the reaction spot is triggered (1) by known information about itsposition relative to the known position of the reaction spots or (2) bysensing a property of the glycerol spot which triggers the delivery offluid via ink-jet printing to the reaction spot. For example, abifurcated fiber optic could excite a fluorescent dye in the reactionspot whose emission is transmitted via the fiber optic cable to anemission filter and a photomultiplier tube. The output of thephotomultiplier tube is amplified, digitized and triggers the printingevent. Alternatively, any optical property of the reaction spot can besensed to trigger the activation of the bioreaction zones.

With any of the above-described sample application techniques or theirequivalents, masking devices may be used to encourage sample applicationdirectly to the microdot. A masking device, such as a template orpattern, may be temporarily placed over dot array or microarray, ormasking materials may be incorporated into the sample applicationequipment. Masking should be understood as optional to the presentinvention.

The assay is preferably contained within a computer-controlled gas flowrate metering system. A supply of inert carrier gas is supplied at highpressure with a regulator to control system pressure to under 50 psig.Typical pressure settings are between 5 to 15 psig. The gas flow usesthe Bernoulli effect to put the aerosol stream toward the target slideto be activated The carrier gas can be any of the following: air,oxygen, nitrogen, helium or argon. The gas flow rate can be between 0.1and 5 L/min to help direct the aerosol from various nozzles.Alternatively, gas flow may be used to carry the liquid sample into thepressure nozzle by Bernoulli effect. It should be understood, however,that the use of these carrier gases is optional to the present methodand apparatus.

For sample application and assay monitoring, the assay system preferablyincludes a computer controlled xy positioner with an independent x and yaxis of movement. The xy positioner translates a removable stage thatcontains an accessible housing for assay chips ready for microdotapplication and assay activation. The removable stage allows transferfrom the arrayer into the sprayer, and subsequently into the incubatorwhile avoiding hands on contact between the operator and the individualslides. The xy positioner has a travel range from 0.1 m by 0.1 m up to10 m by 10 m. Smaller distances of the xy positioner stage travel can beachieved by a linear stepper motor or servomotor. Longer distances canbe achieved by motorized belt drive assemblies with motors that arecontrolled by individual drivers that receive driving signals from themain operating computer. A velocity of 0.1 to 20 in/sec can be achievedfor rapid translation of slides under the aerosol.

Specifically, the assay method may involve dissolving a fluorogenicsubstrate in a volume of DMSO at 10 μM to 1 M concentration. A 0.1-1volume of glycerol is then mixed with the substrate DMSO solution. Thissolution is delivered to a glass substrate by micromanipulation of asmall diameter metal or plastic probe. The microdot can release itsvolatile DMSO component at room temperature. A mask is placed over theglass substrate to cover at least a portion of the areas among themicrodots. A microspray of biological fluid is delivered through themask to the microdots on the surface. The microdot array is incubatedfrom 0 to 180 mins at 4-37° C. in 0 to 100% humidity. Conversion ofsubstrate is detected in the microdot by excitation (350-400 nm) andemission above 420 nm using any available detection means.

It should be borne in mind that, in the practice of the presentinvention, the substrate need not start out as chromogenic orfluorogenic if it can be made so later in the process of assaying thesample. A second spray containing a reporter substrate is described tothis end in Example 1 below.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients, reaction conditions and soforth used in the specification and claims are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numberical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical values, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

Also, it should be understood that any numerical range recited herein isintended to include all sub-ranges subsumed therein. For example, arange of “1 to 10” is intended to include all sub-ranges between andincluding the recited minimum value of 1 and the recited maximum valueof 10, that is, having a minimum value equal to or greater than 1 and amaximum value of equal to or less than 10.

In additional aspects, the present invention provides a method oftransferring molecules of interest from an electrophoretic polymer gelto a MALDI target plate comprising the steps of:

-   -   (i) providing an electrophoretic gel containing one or more        molecules of interest;    -   (ii) replacing water within the electrophoretic gel with a        cosolvent mixture;    -   (iii) positioning a pin over the gel and penetrating the gel        with the pin;    -   (iv) energizing the pin to deplete the gel in a region        surrounding the one or more molecules of interest, causing the        cosolvent mixture to surround the one or more molecules of        interest;    -   (v) lifting the pin out of the gel, the pin carrying a drop of        the cosolvent mixture containing the one or more molecules of        interest; and    -   (vi) contacting a MALDI target plate with the pin, the        contacting causing the drop of cosolvent mixture containing the        one or more molecules of interest to be deposited on the MALDI        target plate. A schematic diagram illustrating this aspect is        shown in FIG. 24.

Any method of gel electrophoresis separation can be used, so long as itprovides adequate separation of the molecules of interest in the initialsample.

After separation of the molecules of interest, the gel is incubated in awater and polyol cosolvent mixture. Preferably, the polyol is glycerol,although other polyols having a viscosity, surface tension and vaporpressure similar to that of glycerol can be used. These propertiesprovide the cosolvent mixture with the ability to adhere to the pin whenthe pin contacts the gel to remove a drop of fluid, and also permit thetransfer of the drop to the target plate by adhesion of the drop to thetarget plate. These properties also ensure that a drop of the cosolventmixture containing the one or more molecules of interest will maintainits position on the MALDI target plate, without substantial evaporation.

The cosolvent mixture is preferably 10% to 90% by volume polyol, usually15% to 35% polyol. Water in the electrophoretic gel is replaced by thecosolvent mixture by incubating the gel in the mixture for a period ofbetween 15-120 minutes.

The pin is a hollow or solid tipped pin as used in a standardmicroprinting or pin printing apparatus, as will be known to one skilledin the art. Preferably, the diameter of the pin at the tip (at the pointof contact with the gel) is between 50 microns to 500 microns.Typically, the pin will be positioned over the gel in an area of highprotein (or other molecule) concentration, as indicated by dye staining.

The pin is energized by ultrasound or other means. If ultrasound isused, the energy of the ultrasound vibration is between 0.1 and 5 wattsper square centimeter, with a frequency between 10 kilohertz to 1megahertz. The pin is energized for 10 seconds to 120 seconds.

Energizing the pin results in the depletion or collapse of the polymergel in a region surrounding the pin. Cosolvent mixture and other fluidsfrom the gel flow into the collapsed region and surround the one or moremolecules of interest in the region surrounding the pin. Due to theviscosity, surface tension and vapor pressure of the cosolvent mixture,when the pin is lifted out of the gel the pin will carry a drop of thecosolvent mixture containing the one or more molecules of interest. Thisdrop can then be deposited on a MALDI target plate, by contacting theplate with the pin. The drop of cosolvent mixture containing the one ormore molecules of interest will adhere to and be deposited on the MALDItarget plate. The pin can then be washed in a submersion bath and thesteps repeated one or more times to deposit a plurality of drops on thetarget plate. The word “drops” and “spots” will be used hereininterchangeably to refer to the samples deposited on the MALDI targetplate.

The drops of cosolvent mixture containing the molecules of interestdeposited on the MALDI target plate by the methods of the presentinvention will be between 1 to 2000 nanoliters in size, more preferablybetween 1 to 100 nanoliters in size. The diameter of the drops will bebetween 50 and 500 microns, and the density of drops on the target plateis between 100 and 1000 drops per square centimeter.

After the drops are deposited on the target plate, a reagent chosen toreact with the molecules of interest is deposited on the target plate,such that the reagent contacts the deposited drops containing the one ormore molecules of interest and the reaction proceeds. The reagent can bedeposited by standard methods known in the art, including but notlimited to aerosol deposition, microprinting, pin printing, positivedisplacement pipetting and piezo printing.

The one or more molecules of interest include, but are not limited to,proteins, peptides, DNA, RNA, nucleotides, enzymes, amino acids,substrates, catalysts, salts, buffers, cofactors, reaction-alteredchemical compounds, a member of a combinatorial library of chemicalcompounds, a component of a drug screening reaction and combinationsthereof.

After the reactions have run to completion, the target plate with thedeposited drops is prepared for MALDI mass spectrometry analysis bydrying the deposited drops and coating the target plate with a MALDImatrix.

In a further aspect, the present invention provides a method of rungchemical reactions on a MALDI target plate comprising: depositing dropsof reactants on the target plate; depositing a reagent on the targetplate such that the reagent contacts the deposited drops; and allowingthe chemical reaction to proceed. The drops and the reagent aredeposited on the target plate by any suitable means known in the art,including, but not limited to aerosol deposition, microprinting, pinprinting, piezo printing, and positive displacement pipetting.Preferably, the volume of each deposited drop is between 1 to 2000nanoliters, and also preferably, the density of drops on the targetplate is between 100 and 1000 drops per square centimeter. Suitablereagents and reactants include, but are not limited to, proteins,peptides, DNA, RNA, nucleotides, enzymes, amino acids, substrates,catalysts, salts, buffers, cofactors, reaction-altered chemicalcompounds, a member of a combinatorial library of chemical compounds, acomponent of a drug screening reaction and combinations thereof.

In yet a further aspect, the present invention provides a method ofpreparing a sample for MALDI mass spectrometry analysis comprising thesteps of:

-   -   (i) providing a target plate having liquid drops of sample;    -   (ii) drying the target plate to remove solvents from the sample        drops;    -   (iii) depositing a MALDI matrix onto the dry target plate;    -   (iv) humidifying the target plate; and    -   (v) subjecting the target plate to MALDI mass spectrometry for        analysis of the sample drops. These aspects of the invention are        illustrated in the schematic diagram of FIG. 25.

The liquid sample drops comprise the reaction product of one or morereactants and one or more reagents. Suitable reactants and reagentsinclude, but are not limited to, proteins, peptides, DNA, RNA,nucleotides, enzymes, amino acids, substrates, catalysts, salts,buffers, cofactors, reaction-altered chemical compounds, a member of acombinatorial library of chemical compounds, a component of a drugscreening reaction and combinations thereof.

Preferably, the liquid sample drops have a volume of between 1-2000nanoliters, and the density of liquid sample drops on the target plateis between 100 and 1000 drops per square centimeter.

The drying step is effected by any suitable method, such as vacuumdrying or air drying.

Preferably, the matrix is deposited by aerosol deposition means, as isknown in the art. Also preferably, the matrix is deposited in a layer ofless than 50 microns in thickness, and less than 10 microliters ofmatrix is deposited per every 5 square centimeters of target plate. Oneor more layers can be deposited to achieve the desired thickness.

The matrix material comprises volatile solvents and a supersaturatedconcentration of matrix compounds. Matrix materials are well known inthe art, and include but are not limited to the compositions listed inTables 2 and 3, as described in Matrix Materials for Matrix-AssistedLaser Desorption Ionization-Time of Flight (MALDI-TOF): Guidelines forSelection (APPLIED BIOSYSTEMS—Voyager-DE-Biospectrometry Workstations).TABLE 2 Commonly Used MALDI Matrices For Positive Ion Modeα-cyano-4-hydroxycinnarnic acid or HCCA a. 5-10 mg/ml of matrix -insolution of 50:50 or 70:30 acetonitrile/ aqueous 0.1% Trifluoroaceticacid b. 5-10 mg/ml of matrix in solution of 60% ethanol/36%acetonitrile/ 4% water 2,5-Dihydroxybenzoic acid or DHB a. 5-10 mg/ml ofmatrix in solution of 10% aqueous ethanol b. 5-10 mg/ml of matrix insolution of 50:50 or 70:30 acetonitrile/ aqueous 0.1% Trifluoroaceticacid 3,5-Dimethoxy-4-hydroxycinnamic acid or sinapinic acid a. 5-10mg/ml of matrix in solution of 50:50 acetonitrile/aqueous 0.1%Trifluoroacetic acid 2-(4-hydroxyphenylazo)benzoic acid or HABA a. 1.5mg/ml of matrix in solution of 50:50 acetonitrile/water or 40:40:20acetonitrile/methanol/water

TABLE 3 Commonly Used MALDI Matrices For Negative Ion Modeα-cyano-4-hydroxycinnamic acid or HCCA a. 5-10 mg/ml of matrix insolution of 50:50 or 70:30 acetonitrile/ aqueous 0.1% Trifluoroaceticacid b. 5-10 mg/ml of matrix in solution of 60% ethanol/36%acetonitrile/ 4% water 2,5-Dihydroxybenzoic acid or DHB a. 5-10 mg/ml ofmatrix in solution of 10% aqueous ethanol b. 5-10 mg/ml of matrix insolution of 50:50 or 70:30 acetonitrile/ aqueous 0.1% Trifluoroaceticacid Hydroxypicolinic acid or HPA a. 25 mg/ml of matrix in solution of50% acetonitrile with 2.5 mg/ml of diammonium tartrate or citrate6-aza-2-thiothymine a. 10 mg/ml of matrix in solution of 50:50acetonitrile/20 mM ammonium citrate

The humidification step is carried out in a standard scientifichumidification chamber at a relative humidity of 40% to 80%, at atemperature of 22° C. to 37° C. for a period of 10 minutes to 120minutes. Care must be taken to ensure that humidification occurs withoutsubstantial deposition of water droplets on the MALDI matrix coating.Humidification causes the sample drops to become semi-solid and theconstituents of the matrix are able to admix with the reaction productsin the sample drops.

The matrix formed using the methods of the present invention providesdetection of reaction products in the sample drops in the range of 1 to50 femtomoles per sample drop. After deposition of the matrix, due tothe ultrasmall drop size salt ions in the sample drops are able todiffuse away from the reaction products into the surrounding matrix,thus providing outstanding crystals for the subsequent MALDI MSanalysis. Methods of the present invention can be used, for example, todetect activity of the drug on the molecule where the reactant is abiological molecule and the reagent is a drug.

In a further aspect, the present invention provides a method ofdepositing one or more layers of a MALDI matrix on a target platecomprising:

-   -   (i) providing a target plate having samples thereon;    -   (ii) aerosolizing the matrix; and    -   (iii) spraying the aerosolized matrix on the target plate while        moving the target plate.

The matrix can aerosolized by standard methods known in the art, such aswith an ultrasonic nozzle or a spray nozzle. Preferably, an ultrasonicnozzle is used, as it permits deposition of very small droplets and avery thin layer of matrix. The gas flow rate of the ultrasonic nozzle orspray nozzle is between 0.1 to 5 microliters per minute, more preferably0.5 to 2.0 microliters per minute. If an ultrasonic nozzle is used,preferably the energy of nozzle is set to between 0.1 to 2 watts, morepreferably 0.5 to 1 watt. Preferably, the matrix is deposited at athickness of less than 50 microns, and the target plate is moved at arate of 0.1 to 5 inches per second, more preferably 0.5 to 2 inches persecond, in a linear direction.

EXAMPLES Example 1

A microarray of glycerol loci is created in which each glycerol spot (50to 250 micrometers in diameter with a 50 to 500 micrometer space betweenspots) contains a single molecular species “A” such as a protein orprotein fragment; a synthetic peptide; a small organic molecule; anucleic acid sequence or a synthetic polymer. This species resides at aconcentration between 10 picomolar and 10 millimolar within the spot. A1″×3″ glass slide is configured to yield over 10 cm² of arrayed glycerolloci at 100 to 1000 spots/cm².

An aerosol containing an enzyme (at 1 picomolar to 10 millimolar) isdelivered to the array, whereby droplets of the enzyme fuse with thereaction spots while droplets that hit the slide quickly evaporate. Fora period of time, the enzyme is allowed to come into binding equilibriumwith the chemical species, typically 1 to 60 mins. A second spraycontains a reporter system to detect the enzyme activity. Such areporter system may consist of a fluorogenic substrate; a chromogenicsubstrate; a cofactor along with a detection system for cofactorconversion; or a cofactor and a substrate, whereby cofactor conversionproduces a detectable signal. This reporter system can be delivered atconcentrations from 10 nM to 100 mM with a fluorogenic substrate.

If the molecular species “A” binds the active site or an allostericposition of the enzyme “E”, then the activation of the reporter system(e.g., conversion of a fluorogenic substrate) is prevented. Reactionzones that produce no reporter signal or reduced reporter signal arethen identified as inhibitors of the enzyme. The inhibitor effect may bedue to the molecular species “A” acting as a competitive inhibitor; asuicide inhibitor; a noncompetitive inhibitor; an allosteric modulator;a complexation agent against the substrate; an antagonist of cofactorbinding; a complexation agent against the cofactor or an uncompetitiveinhibitor.

Example 2

A microarray is set up to analyze binding events between antigens andantibodies. Initially, a polyclonal or monoclonal antibody is chemicallylinked to a colloidal object, such as colloidal gold (10 nM to 200 nM)or latex bead (10 nM to 200 nM). The latex beads with covalently linkedantibodies are added to glycerol to create a suspension prepared formicroarraying. The glycerol may also contain an unlinked polyclonal ormonoclonal antibody against a particular antigen of detection. Theconcentration of the antibody covalently linked to the bead may rangefrom 1 to 10,000 sites per square micron of the bead. The concentrationof the free antibody in solution can range from 0 to 100 μM.

An array of spots is generated (spot size of 50 to 250 micrometerdiameter with a 50 to 500 micrometer space between spots). Each spot hasa unique composition whereby colloidal objects with linked antibody aremaintained at a single concentration and the quenching free antibody ismaintained at increasing concentration in a series of spots. Abiological sample containing the antigen of detection is delivered viaspray mechanism to the array. In each reaction spot, the quenchingantibody binds the antigen (a faster reaction since the free antibodieshave greater Brownian motion in comparison to the antibodies bound tothe beads). If the concentration of the antigen exceeds theconcentration of the quenching antibody, then free antigen remains andis available for binding to the beads. Beads will undergoantigen-mediated agglutination. The agglutinated colloidal gold or latexbeads can be quantified by computer-assisted imaging of the aggregates,enhanced light scattering by aggregates or reduced light transmission byaggregates. Through use of known increasing concentrations of quenchingantibodies in a series of spots, the concentration of antigen can beestimated to be equivalent to the minimum concentration of quenchingantibody preventing agglutination.

Example 3

Detection of an antigen in a biological fluid is accomplished with anon-fluorescent species of unknown concentration. A series of glyceroldroplets is arrayed with each containing a fluorescent species with abinding site. The reporter molecular would be under 30 kDa. Such areporter molecule would be a GFP-ScFv chimeric protein where the singlechain Fv fragment has high binding affinity for the antigen to bedetected.

The antigen to be detected is sprayed onto the array surface and issufficiently large (greater than 5 kDa) causing a significant change inthe total molecular weight of the detector molecule/antigen complex. Thedetector molecule/antigen complex is analyzed by the polarization of thefluorescence emission of the complex which is greater than that of thereporter molecule alone when the system is excited with polarized light.

Example 4

Well characterized for over 30 years, serine proteases of thecoagulation pathway in plasma were selected as a system in which tomonitor the delivery of complex enzyme fluids to the microassay.Fluorogenic substrates in DMSO/glycerol were assayed to a glass slide.

After spray application, thrombin caused a 65-fold increase influorescence activity after conversion of the boc-VPR-MCA substrate.When deposited on the array, pure plasmin (1 μM) gave an intense signalfor the boc-VLK-MCA substrate (25-fold above background) with detectableactivity on the thrombin and kallikrein substrates. Plasmin has beenreported to have activity on boc-VPR-MCA and Z-FR-MCA with no detectableactivity on tPA and factor Xa substrates (Morita, 1977). Aerosoldeposition of pure tPA (10 μM) to a sensing array caused significantcleavage of the tPA, thrombin and Xa substrates, resulting influorescence that was 18- to 20-fold above background.

The microarray detected the modest activation of the coagulation pathwayin recalcified diluted plasma due to low level factor Xa and thrombinactivity (Butenas, 1997; Rand, 1996). Since corn trypsin inhibitor wasnot present to inhibit factor XIIa (Rand, 1996), moderate activation ofthe contact system and kallikrein generation was detected by conversionof the kallikrein substrate. Recalcified citrated plasma does notgenerate plasmin in excess of the ∀2-antiplasmin and ∀2-macroglobulinconsistent with the lack of conversion of the plasmin substrate. Themicroarray revealed that the conversion of kallikrein substrate occurredfrom the contact activation of prekallikrein to kallikrein, not fromnon-specific Z-FR-MCA substrate cleavage by plasmin.

Addition of plasmin (0.43 μM final concentration) to the dilutedrecalcified citrated plasma overcame inhibitory concentrations of∀2-antiplasmin and ∀2-macroglobulin. This plasmin activity was detectedby a strong signal in the plasmin substrate spot. Plasmin can inactivatefactor Xa (Pryzdial, 1999) and this reduction in factor Xa activity wasobserved in the microassay. Plasmin is also a potent activator of factorXU to factor XUa which can in turn convert prekallikrein to kallilkrein.A high level of kallilkrein substrate conversion was noted in theplasmin treated-plasma beyond that expected from plasmin-mediatedconversion of the kallikrein substrate. Addition of a higher level ofplasmin (2.14-1 μM final concentration) led to significant cleavage ofeach substrate on the array. The microarray revealed thatplasmin-mediated activation of factor XIIa resulted in kallikreinproduction with sufficiently increased intrinsic pathway production offactor Xa to overcome plasmin-mediated factor Xa proteolysis to factorXaa.

Example 5

Programmed cell death (apoptosis) can be triggered by various cellularevents such as calcium influx; oxidative stress; cytoskeletalinterference; inhibitors of protein synthesis; membrane disruption orDNA disruption. Numerous chemicals induce apoptosis in specific celltypes and include receptor ligands (TRAIL, FasL); ceremide-based lipids;taxol; vinblastine; cytochalasin D; topoisomerase inhibitors(etoposide); DNA cross linking agents; protein linase inhibitors andmitochondria permeability agents (betulinic acid, rotenone).

Apoptosis occurs after activation/aggregation of surface receptors. Thebest studied death receptors are TNFR1 (p55 or CD120a) and CD95 (FasR orApo1). Other death receptors include DR3 (Apo3), DR4 and DR5 (TRAIL-R2,Apo2). In cancer therapeutics, soluble TRAIL (Apo2L) can bind itsreceptors DR4 and DR5 to activate apoptosis in transformed cell linesbut not in normal cells. Downstream signaling of death receptors is wellunderstood, but complex. As an example, homotrimeric CD95L binds CD95which undergoes clustering and subsequent binding of a Fas-associateddeath domain (ADD) protein which in turn activates Caspase 8 (FLICE).After oligomerization, Caspase 8 undergoes autoactivation which in turnactivates Caspase 9. Pathways distal to TNF binding TRNR1, Apo3L bindingto DR3, and Apo2L binding DR4 or DRS result in multimerization ofreceptors, adaptors and activation of caspases. Several caspases canproteolytically inactivate poly(ADP-ribose) polymerase (PARP) anddegrade nuclear lamin which are key signatures of apoptosis.

The role of the mitochondria in apoptosis is thought to result from theancient two-billion year old symbiosis that produced eucaryotic cells.Loss of mitochondria integrity disrupts energy (ATP) production,triggers caspase activation and disturbs the redox potential of thecell. In caspase activation, cytochrome c (blocked by apoptosisinhibitor bc1-2) released from the mitochondria can complex with Apaf-1and Procaspase 9 resulting in activation of Caspase 9.

Caspases (cysteinyl aspartate-specific proteases) cleave proteinsubstrates on the carboxyl terminus side of aspartate (P1 position).Positions P2, P3 and P4 also contribute to substrate specificity with P4residues having the largest role in dictating substrate preferencesamong the caspases. A total of 13 distinct caspases have been identifiedso far. Various caspases can cleave a given fluorogenic substrate andthe use of the term “a Caspase 3 substrate” does not imply that othercaspases do not cleave this substrate or that Caspase 3 does not cleaveother substrates. The substrate specificity of caspases has been studiedthrough the synthesis of chromogenic and fluorogenic peptide libraries(Talanian, 1997; Thornberry, 1997). Thornberry used a 60-compoundfluorogenic positional scanning library Ac-X-X-X-Asp-AMC to evaluate thespecificity in brackets of Caspase 1 [WEHD]; Caspase 2 [DEHD]; Caspase 3[DEVD]; Caspase 4 [(W/L)EHD]; Caspase 5 [(W/L)EHD]; Caspase 6 [VEHD];Caspase 7 [DEVD], Caspase 8 [LETD]; Caspase 9 [LEHD] and Granzyme B[IEPD]. Similarly, various peptide aldehydes have been tested forspecificity of inhibition (Garcia-Calvo, 1998) with second order rateconstants >10⁵ M⁻¹s⁻¹. TABLE 1 Inhibitor EETD- VDVAD- DEVD- YVAD- LEHD-VEID- CHO CHO CHO CHO CHO CHO Substrate 16 15 14 13 12 11 Blank VEIDS1 + I6 S1 + I5 S1 + I4 S1 + I3 S1 + I2 S1 + I1 S1 LEHD S2 + I6 S2 + I5S2 + I4 S2 + I3 S2 + I2 S2 + I1 S2 YVAD S3 + I6 S3 + I5 S3 + I4 S3 + I3S3 + I2 S3 + I1 S3 DEVD S4 + I6 S4 + I5 S4 + I4 S4 + I3 S4 + I2 S4 + I1S4 VDVAD S5 + I6 S5 + I5 S5 + I4 S5 + I3 S5 + I2 S5 + I1 S5 IETD S6 + I6S6 + I5 S6 + I4 S6 + I3 S6 + I2 S6 + I1 S6

Example 6

A biological fluid is delivered to the chip surface as an aerosol wherethe fluid is a liquid sample obtained from blood; urine; saliva; biopsy;microbe or microbial preparation; virus or viral preparation; celllysate or cell suspension or a food or agricultural product.Alternatively, the aerosol may be composed of a carrier gas such as airor nitrogen mixed with a sample gas in which are dispersed protein,viral or bacterial particles. The sample is tested for enzymaticactivity where fluorogenic substrates have been arrayed in glycerol MCAsubstrates and enzymes.

Example 7

A suspension of cells, DNA, total RNA or mRNA is delivered to reactionzones arrayed on a microassay chip. Individual reaction zones containPCR primers; reverse transcriptase primers; dye-labeled oligo sequences;nucleic acid bases or fluorescent bases and enzymes such as reversetranscriptase; DNA polymerase; RNAse; DNAse; heat stable DNA polymeraseor cleavase enzyme. The chip is subjected to heat cycles for PCR ornucleic acid synthesis or fluorescence tag incorporation or fluorescenceactivation of quenched entities via sequence dependent reactions.Subsequent detection can involve energy transfer between two independentfluorescent probes brought into proximity by a sequence dependentreaction dequencing of quenched molecules due to a sequence dependentreaction. Applications can include phenotypic analysis of mRNA species,genotypic analysis of DNA species and detection of single nucleotidepolymorphisms (SNPs).

Example 8

Microarrays have numerous applications in protease engineering andproteomics. A constant P1 positional scanning library of fluorogenicpeptides with 19 different amino acids at the P2, P3 or P4 position (57sublibraries) (Backes, 2000) can be accommodated by <1 cm² of microarraywith minimal usage of reagents. An entire scanning fluorogenic library(Harris, 2000) with 19 to 20 different amino acids in the P1-P4positions (<1200 spots) could be accommodated on a 1″×3″ slide wellwithin the capability of glycerol spotting and aerosol depositiontechnology. In this example, a single protease is applied to individualfluorogenic substrates arrayed on the chip from a positional scanninglibrary. Conversion of substrates and substrate specificity can bedetermined on a single microassay chip. Also, a combinatorial library offluorogenic peptides where the identity of each amino acid in eachposition is well-established can be employed on a microassay chip.

Example 9 Removal of Sample from Gel and Transfer to MALDI Target Plate

For direct detection, 1 μg of purified human fibrinogen per lane isheated to 95° C. for 5 min in SDS running buffer (Tris-EDTA, pH 8.0, 2%vol/vol SDS, 8 M urea). The protein sample is run on a one dimensional4-15% gradient polyacrylamide gel and then stained with commassie bluedye for 60 min, the position of the band noted, and the gel destainedwith NH4/HC03/50% acetonitrile, followed by incubation in 30% by vol.glycerol for 120 min at room temperature with gentle orbital mixing. Thegel is briefly rinsed with distilled water and a pin is positioned abovethe location of the protein band, and submerged halfway into the gel andenergized by coupling to an ultrasonic transducer (30 kHz, Sonicare) for20 sec. The pin is slowly removed from the gel and placed in contactwith the metal MALDI target surface by contact printing (GeneMachinesAscent). A single pin protocol using stealth SMP15XB pins from Telechem(Sunnyvale, Calif.) is used to print a 500 um spot, onto the SS plateusing the OmniGrid Accent microarrayer from Genemachines (now part ofGenomic Solutions, Ann Arbor, Mich.). For proteolytic digestion, thefibrinogen is then activated with 10 uM of human thrombin (50 mM SodiumCitrate, 0.2 M NaCl, 0.1% PEG-8000 and pH 6.5). Human thrombin isdelivered to the array via a 120 kHz ultrasonic nozzle (Sonotek, Milton,N.Y.). The sample is aerosolized at a liquid flow rate of 400 nl/s intothe nozzle using a UMPII flow pump (World Precision Instruments,Saratoga, Fla.) and sheathed with a carrier air flow of 2.3 L/min. Afteraerosol deposition for 2 or 4 s, the SS plate with the reactions isincubated at 37° C. for 4 hrs. The reaction is then stopped by overnightextraction of glycerol leaving behind fine crystals of the cleavageproduct of fibrinogen in presence of thrombin: fibrinopeptide A (Avg.MW: 1536.57 Da) and fibrinopeptide B (Avg. MW: 1569.60 Da). The cleavageproducts are detected using the Voyager DE-PROmatrix-assisted-laser-desoprtion-ionization time-of-flight (MALDI TOF)mass spectrometer (MS) from Applied Biosystems (Foster City, Calif.).For detection purposes the plate is coated with a thin film of3,5-Dimethoxy-4-hydoxycinnamic acid (sinapinic acid) in 50% ethanol.This is a typical positive ion mode matrix used for MALDI TOF MS. The MSis used in the positive linear mode with the laser source maintained ata constant power for al detection. The peaks for fibrinopeptide A and Bare obtained at their respective m/z mass ranges (data not shown).

Example 10 High Throughput Screening

The ability to run nanoliter reactions using libraries printed on flatsurfaces allows for the use of MALDI mass spectrometry to probe HTSreactions. The feasibility of running label-free reactions by printingfibrinogen solutions on a metal MALDI target, followed by aerosoldeposition of thrombin to release fibrinopeptides A and B, has beendemonstrated. The glycerol in the reaction was then removed, and MALDImatrix was deposited to result in a thin film suitable (FIG. 26A) for MSdetection of FPA/FPB. Benzamidine in the nanoliter reaction completelyinhibited the reaction (FIG. 26B). These sensitivity of MALDI to detectcalibrants and converted substrates in the range of 3 to 300 nM is idealfor the typical reaction conditions needed in HTS assays.

A 8×12 array of reactions were printed using an OmniGrid microarrayercontaining varying concentrations of fibrinogen and a fibrinopeptide MSsatandard. Certain reactions contained 1 mM benzamidine. The array wasactivated with thrombin and incubated under humidity (top row, A), theglycerol was removed (middle row, A), and then coated with MALDI matrix(bottom row, A). Individual reactions were then subjected to MALDI foranalysis of fibrinopeptides A and B. A large signal was seen at 1, 10,and 100 ug/ml of fibrinogen (3, 30 and 300 nM) treated with thrombin,but not in screening reactions containing benzamidine (B). Conditions ofthe MALDI MS were: Voyager-DE PRO (sinapinic acid matrix),Glu1-Fibrinopeptide B (1 ug/ml) control, Reflector Mode, PositivePolarity, 1000-2000 Da Acquisition Range, 20000 V Accelerating voltage,2033 laser intensity (20.0 Hz; 100/spectrum), Extraction delay time: 150nsec, Grid voltage: 75%, Guide wire voltage: 0.002%.

Example 11 Detection of Protein/Enzyme Interaction

Plasminogen depleted human fibrinogen (MW:330 kDa) was purchased fromEnzyme Research Labs (South Bend, Ind.). It was aliquoted in themanufacturer recommended buffer of 10 mM Phosphate, 20 mM Citrate, 0.15M NaCl (pH 7.4) at a concentration of 36 mg/ml. Six fold dilutions—18mg/ml, 9 mg/ml, 4.5 mg/ml, 3 mg/ml, 1.5 mg/ml and 0.75 mg/ml of 1:1plasminogen depleted fibrinogen and glycerol were performed andaliquoted separately. Twelve replicates of each solution were thenprinted in on to a single blank stainless steel (SS) mass spectrometryplate from Applied Biosystems (Foster City, Calif.). A single pinprotocol using stealth SMP15XB pins from Telechem (Sunnyvale, Calif.)was used to print 500 um spots, onto the SS plate using the OmniGridAccent microarrayer from Genemachines (now part of Genomic Solutions,Ann Arbor, Mich.). The spot to spot spacing was 1000 um.

The fibrinogen was then activated with 10 uM of human thrombin (50 mMSodium Citrate, 0.2 M NaCl, 0.1% PEG-8000 and pH 6.5). Human thrombinwas delivered to the array via a 120 kHz ultrasonic nozzle (Sonotek,Milton, N.Y.). The sample was aerosolized at a liquid flow rate of 400nl/s into the nozzle using a UMPII flow pump (World PrecisionInstruments, Saratoga, Fla.) and sheathed with a carrier air flow of 2.3L/min. After aerosol deposition for 2 or 4 s, the SS plate with thereactions was incubated at 37° C. for 4 hrs. The reaction was thenstopped by overnight extraction of glycerol leaving behind fine crystalsof the cleavage product of fibrinogen in presence, r thrombin:fibrinopeptide A (Avg. MW: 1536.57 Da) and fibrinopeptide B (Avg. MW:1569.60 Da).

The cleavage products were detected using the Voyager DE-PROmatrix-assisted-laser-desorption-ionization time-of-flight (MALDI TOF)mass spectrometer (MS) from Applied Biosystems (Foster City, Calif.).For detection purposes the plate was coated with a thin film of3,5-Dimethoxy-4-hydoxycinnamic acid (sinapinic acid) in 50% ethanol.This is a typical positive ion mode matrix used for MALDI TOF MS. The MSwas used in the positive linear mode with the laser source maintained ata constant power for al detection. The peaks were for fibrinopeptide Aand B were obtained at their respective m/z mass ranges. Mass spectrumdata is presented in FIGS. 27 A-O for the 18 mg/ml, 3 mg/ml and 0.75mg/ml samples.

Although the invention has been described above with reference toparticular materials and methods, the invention is only to be limitedinsofar as is set forth in the accompanying claims.

1. A method of transferring molecules of interest from anelectrophoretic polymer gel to a MALDI target plate comprising the stepsof: (i) providing an electrophoretic gel containing one or moremolecules of interest; (ii) replacing water within the electrophoreticgel with a cosolvent mixture; (iii) positioning a pin over the gel andpenetrating the gel with the pin; (iv) energizing the pin to deplete thegel in a region surrounding the one or more molecules of interest,causing the cosolvent mixture to surround the one or more molecules ofinterest; (v) lifting the pin out of the gel, the pin carrying a drop ofthe cosolvent mixture containing the one or more molecules of interest;and (vi) contacting a MALDI target plate with the pin, the contactingcausing the drop of cosolvent mixture containing the one or moremolecules of interest to be deposited on the MALDI target plate.
 2. Themethod of claim 1, wherein the viscosity, surface tension and vaporpressure of the cosolvent mixture cause the drop of cosolvent mixturecontaining the one or more molecules of interest in the gel to adhere tothe pin.
 3. The method of claim 1, wherein the viscosity, surfacetension and vapor pressure of the cosolvent mixture cause the drop ofcosolvent mixture containing the one or more molecules of interest to betransferred to and adhere to the MALDI target plate from the pin.
 4. Themethod of claim 1, wherein the viscosity, surface tension and vaporpressure of the cosolvent mixture cause the drop of cosolvent mixturecontaining the one or more molecules of interest to maintain itsposition on the MALDI target plate, without substantial evaporation. 5.The method of claim 1, wherein the cosolvent mixture is a water andglycerol mixture of 10% to 90% by volume glycerol.
 6. The method ofclaim 1, wherein the cosolvent mixture is a water and polyol mixture. 7.The method of claim 1, wherein the energizing of the pin is effected byultrasound vibration.
 8. The method of claim 7, wherein the energy ofthe ultrasound vibration is between 0.1 and 5 watts per squarecentimeter.
 9. The method of claim 7, wherein the frequency of theultrasound vibration is between 10 kilohertz to 1 megahertz.
 10. Themethod of claim 1, wherein the pin is energized for 10 seconds to 120seconds.
 11. The method of claim 1, wherein the diameter of the pin atthe tip is between 50 microns to 500 microns.
 12. The method of claim 1,wherein the drop of cosolvent mixture containing the one or moremolecules of interest is between 1 to 2000 nanoliters in size.
 13. Themethod of claim 1, wherein the drop of cosolvent mixture containing theone or more molecules of interest is between 1 to 100 nanoliters insize.
 14. The method of claim 1, wherein the diameter of the dropdeposited on the MALDI target plate is between 50 and 500 microns. 15.The method of claim 1, further comprising the step of washing the pin ina submersion bath and repeating the steps of claim 1 one or more timesto deposit a plurality of drops on the target plate.
 16. The method ofclaim 15, wherein the density of drops on the target plate is between100 and 1000 drops per square centimeter.
 17. The method of claim 15,further comprising the steps of depositing a reagent on the targetplate, such that the reagent contacts the deposited drops of cosolventmixture containing the one or molecules of interest; and allowing thereagent to react with the one or molecules of interest in the depositeddrops.
 18. The method of claim 17, wherein the depositing of the reagentis effected by a means selected from the group consisting of aerosoldeposition, microprinting, pin printing, positive displacement pipettingand piezo printing.
 19. The method of claim 1, wherein the one or moremolecules of interest are selected from the group consisting ofproteins, peptides, DNA, RNA, nucleotides, enzymes, amino acids,substrates, catalysts, salts, buffers, cofactors, reaction-alteredchemical compounds, a member of a combinatorial library of chemicalcompounds, a component of a drug screening reaction and combinationsthereof.
 20. The method of claim 1, wherein the water in theelectrophoretic gel is replaced by the cosolvent mixture by incubatingthe gel in the cosolvent mixture for a period of between 15-120 minutes.21. The method of claim 15, further comprising preparing the targetplate with the deposited drops for MALDI mass spectrometry analysis bydrying the deposited drops and coating the target plate with a MALDImatrix.
 22. A method of running chemical reactions on a MALDI targetplate comprising: depositing drops of reactants on the target plate;depositing a reagent on the target plate such that the reagent contactsthe deposited drops; and allowing the chemical reaction to proceed. 23.The method of claim 22, wherein the drops are deposited on the targetplate by a means selected from the group consisting of pin printing,piezo printing, microprinting and positive displacement pipetting. 24.The method of claim 22, wherein the reagent is deposited on the targetplate by a means selected from the group consisting of aerosoldeposition, microprinting, pin printing, piezo printing and positivedisplacement pipetting.
 25. The method of claim 22, wherein the volumeof each deposited drop is between 1 to 2000 nanoliters.
 26. The methodof claim 22, wherein the density of drops on the target plate is between100 and 1000 drops per square centimeter.
 27. The method of claim 22,wherein the reagents and reactants are selected from the groupconsisting of proteins, peptides, DNA, RNA, nucleotides, enzymes, aminoacids, substrates, catalysts, salts, buffers, cofactors,reaction-altered chemical compounds, a member of a combinatorial libraryof chemical compounds, a component of a drug screening reaction andcombinations thereof.
 28. A method of preparing a sample for MALDI massspectrometry analysis comprising the steps of: (i) providing a targetplate having liquid drops of sample; (ii) drying the target plate toremove solvents from the sample drops; (iii) depositing a MALDI matrixonto the dry target plate; (iv) humidifying the target plate; and (v)subjecting the target plate to MALDI mass spectrometry for analysis ofthe sample drops.
 29. The method of claim 28, wherein the liquid sampledrops comprise the reaction product of one or more reactants and one ormore reagents selected from the group consisting of proteins, peptides,DNA, RNA, nucleotides enzymes, amino acids, substrates, catalysts,salts, buffers, cofactors, reaction-altered chemical compounds, a memberof a combinatorial library of chemical compounds, a component of a drugscreening reaction and combinations thereof
 30. The method of claim 28,wherein the liquid sample drops have a volume of between 1-2000nanoliters.
 31. The method of claim 28, wherein the density of liquidsample drops on the target plate is between 100 and 1000 drops persquare centimeter.
 32. The method of claim 28, wherein the drying stepis effected by vacuum drying or air drying.
 33. The method of claim 28,wherein the matrix is deposited by aerosol deposition.
 34. The method ofclaim 33, wherein the matrix is deposited in a layer less than 50microns in thickness.
 35. The method of claim 33, wherein less than 10microliters of matrix is deposited per every 5 square centimeters oftarget plate.
 36. The method of claim 28, wherein the matrix comprisesvolatile solvents and a supersaturated concentration of matrixcompounds.
 37. The method of claim 28, wherein said humidification stepis carried out at a relative humidity of 40% to 80%, at a temperature of22° C. to 37° C. for a period of 10 minutes to 120 minutes, withoutsubstantial deposition of water droplets on the MALDI matrix coating.38. The method of claim 28, wherein after the humidifying step thesample drops become semi-solid and the constituents of the matrix areable to admix with the reaction products in the sample drops.
 39. Themethod of claim 28, wherein the matrix formed provides detection ofreaction products in the sample drops in the range of 1 to 50 femtomolesper sample drop.
 40. The method of claim 28, wherein after deposition ofthe matrix salt ions in the sample drops diffuse away from the, reactionproducts into the surrounding matrix.
 41. The method of claim 28,wherein the reactant is a biological molecule and the reagent is a drug,for use in detecting activity of the drug on the molecule.
 42. A methodof depositing one or more layers of a MALDI matrix on a target platecomprising: (i) providing a target plate having samples thereon; (ii)aerosolizing the matrix; and (iii) spraying the aerosolized matrix onthe target plate while moving the target plate.
 43. The method of claim42, wherein the matrix is aerosolized by the use of an ultrasonic nozzleor spray nozzle.
 44. The method of claim 43, wherein the gas flow rateof the ultrasonic nozzle or spray nozzle is between 0.5 to 2.0microliters per minute.
 45. The method of claim 44, wherein the energyof the ultrasonic nozzle is between 0.5 to 1 watt per square centimeter.46. The method of claim 42, wherein the matrix is deposited at athickness of less than 50 microns.
 47. The method of claim 42, whereinthe target plate is moved at a rate of 0.5 to 2 inches per second.